This invention relates to Bacillus strains useful for the expression and secretion of desired polypeptides (as used herein, "polypeptide" means any useful chain of amino acids, including proteins).
Bacillus strains have been used as hosts to express heterologous polypeptides from genetically engineered vectors. The use of a Gram positive host such as Bacillus avoids some of the problems associated with expressing heterologous genes in Gram negative organisms such as E. coli. For example, Gram negative organisms produce endotoxins which may be difficult to separate from a desired product. Furthermore, Gram negative organisms such as E. coli are not easily adapted for the secretion of foreign products, and the recovery of products sequestered within the cells is time consuming, tedious, and potentially problematic. In addition, Bacillus strains are non-pathogenic and are capable of secreting proteins by well-characterized mechanisms.
A general problem in using Bacillus host strains in expression systems is that they produce large amounts of proteases which can degrade heterologous polypeptides before they can be recovered from the culture media. The production of the majority of these proteases occurs at the end of the exponential growth phase. At this time, conditions of nutrient deprivation exist and the cells are preparing for sporulation. The two major extracellular proteases are an alkaline serine protease (subtilisin), the product of the apr gene, and a neutral metalloprotease, the product of the npr gene. Secretion of these proteases occurs into the medium, whereas the major intracellular serine protease, Isp-I, is produced within the cells. Other investigators have created genetically altered Bacillus strains that produce below normal levels of one or more of these three proteases. These strains still produce high enough levels of protease to cause the degradation of heterologous gene products prior to purification.
Stahl et al. (J. Bact., 1984, 158:411) disclose a Bacillus protease mutant in which the chromosomal subtilisin structural gene was replaced with an in vitro derived deletion mutation. Strains carrying this mutation had only 10% of the wild-type extracellular production of protease activity. Yang et al. (J. Bact., 1984, 160:15) disclose a Bacillus protease mutant in which the chromosomal neutral protease gene was replaced with a gene having an in vitro derived deletion mutation. Fahnestock et al. (WO 86/01825) describe the construction of Bacillus strains lacking subtilisin activity by replacing the native chromosomal gene sequence with a partially homologous DNA sequence containing an inserted inactivating segment. Kawamura et al. (J. Bact., 1984, 160:442) disclose Bacillus strains carrying lesions in the npr and apr genes. These strains express less than 4% of the extracellular protease activity levels observed in wild-type strains. Koide et al. (J. Bact., 1986, 167:110) disclose the cloning and sequencing of the isp-1 gene and the construction of an Isp-1 negative mutant by chromosomal integration of an artificially deleted gene.
Sloma et al., 1990 J. Bact. 172:1024-1029, employed B. subtilis deleted for the three major proteases (apr, npr, isp) in order to identify three additional residual proteases (epr, bpr, mpr). Blackburn et al., WO 89/10976 also used sporulation competent apr-, npr- strains to isolate what they alledge to be a residual serine protease (rsp) which lacks amino terminal homology to known bacillus proteases.
Genetically altered strains which are deleted for both the major extracellular protease genes (apr and npr) and three residual protease genes (epr, bpr, mpr) produce virtually undetectable levels of protease activity in standard protease assays. However, a resorufin-labeled casein substrate, can be used to detect minor protease activities which are responsible for degradation of some heterologous polypeptides and proteins.